Radiopharmaceuticals for diagnosing Alzheimer&#39;s disease

ABSTRACT

This invention is directed to the use of radiopharmaceuticals in diagnosing Alzheimer&#39;s disease. In particular the radiopharmaceuticals of the invention are able to pass through the blood-brain barrier and bind to the CCR1 receptor present in brain tissue of patients having Alzheimer&#39;s disease.

This application is a continuation of Ser. No. 09/985,938 filed Nov 6,2001 now U.S. Pat. No. 6,676,926 which claims benefit of U.S.Provisional Application Ser. No. 60/246,299, filed Nov. 6, 2000, thedisclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to novel radiopharmaceuticals useful forthe diagnosis of Alzheimer's disease.

BRIEF DESCRIPTION OF THE BACKGROUND ART

Alzheimer's disease is a severe neurodegenerative disorder, andcurrently about 4 million Americans suffer from this disease. As theaging population continues to grow, this number could reach 14 millionby the middle of next century unless a cure or prevention is found. Atpresent, there is no sensitive and specific premortem test for earlydiagnosis of this disease. Alzheimer's disease is currently diagnosedbased on the clinical observation of cognitive decline, coupled with thesystematic elimination of other possible causes of those symptoms. Theconfirmation of the clinical diagnosis of “probable Alzheimer's disease”can only be made by examination of the postmortem brain. The Alzheimer'sdisease brain is characterized by the appearance of two distinctabnormal proteinaceous deposits in regions of the brain responsible forlearning and memory (e.g., cerebral cortex and hippocampus). Thesedeposits are extracellular amyloid plaques, which are characteristic ofAlzheimer's disease, and intracellular neurofibillary tangles (NFTs),which can be found in other neurodegenerative disorders as well. Amyloidpeptides are typically either 40 or 42 amino acids in length (“Aβ¹⁻⁴⁰”or “Aβ¹⁻⁴²”, respectively) and are formed from abnormal processing of alarger membrane-associated protein of unknown function, the amyloidprecurser protein (“APP”). Oligomeric aggregates of these peptides arethought to be neurotoxic, eventually resulting in synaptic degenerationand neuronal loss. The amount of amyloid deposition roughly correlateswith the severity of symptoms at the time of death.

In the past, there have been several attempts for the design ofradiopharmaceuticals that could be used as diagnostic agents for apremortem diagnosis of Alzheimer's disease.

Bomebroek et al. showed that the amyloid-associated protein serumamyloid P component (SAP), labeled with ¹²³I, accumulates at low levelsin the cerebral cortex, possibly in vessel walls, of patients withcerebral amyloidosis (Bomebroek, M., et al., Nucl. Med. Commun. (1996),Vol. 17, pp. 929-933).

Saito et al. proposed a vector-mediated delivery of ¹²⁵I-labeled Aβ¹⁻⁴⁰through the blood-brain barrier. It is reported that the iodinatedAβ¹⁻⁴⁰ binds Aβ amyloid plaque in tissue sections (Saito, Y., et al.,Proc. Natl. Acad. Sci. USA 1995, Vol. 92, pp. 10227-10231).

U.S. Pat. No. 5,231,000 discloses antibodies with specificity to A4amyloid polypeptide found in the brain of Alzheimer's disease patients.However, a method to deliver these antibodies across the blood-brainbarrier has not been described.

Zhen et al. described modifications of the amyloid-binding dye known as“Congo Red™”, and complexes of these modified molecules with technetiumand rhenium. The complexes with radioactive ions are purported to bepotential imaging agents for Alzheimer's disease (Zhen et al., J. Med.Chem.(1999), Vol. 42, pp. 2805-2815). However, the potential of thecomplexes to cross the blood-brain barrier is limited.

A group at the University of Pennsylvania in the U.S.A. (Skovronsky, M.,et al., Proc. Natl. Acad. Sci. 2000, Vol. 97, pp. 7609-7614) hasdeveloped a fluorescently labeled derivative of Congo Red that is brainpermeable and that non-specifically binds to amyloid materials (that is,peptides in β-pleated sheet conformation). This compound would need tobe radiolabeled and then run through pre-clinical screens forpharmacokinetics and toxicity before clinical testing.

Klunk et al. reported experiments with a derivative of Congo Red™,Chrysamine G (CG). It is reported that CG binds synthetic β-amyloid wellin vitro, and crosses the blood-brain barrier in normal mice (Klunk etal., Neurobiol. Aging (1994), Vol. 15, No. 6, pp. 691-698).

Bergström et al. presented a compound labeled with iodine-123 as apotential radioligand for visualization of M₁ and M₂ muscarinicacetylcholine receptors in Alzheimer's disease (Bergström et al., Eur.J. Nucl. Med. (1999), Vol. 26, pp. 1482-1485).

Recently, it has been discovered that certain specific chemokinereceptors are upregulated in the brains of patients with Alzheimer'sdisease (Horuk, R. et al., J. Immunol. (1997), Vol. 158, pp. 2882-2890);Xia et al., J. NeuroVirol. (1999), Vol. 5, pp. 32-41). In addition, ithas been shown recently that the chemokine receptor CCR1 is upregulatedin the brains of patients with advanced Alzheimer's disease and absentin normal-aged brains (Halks-Miller et al, CCR1 Immunoreactiviy inAlzheimer's Disease Brains, Society for Neuroscience Meeting Abstract,#787.6, Volume 24, 1998). Antagonists to the CCR1 receptor and their useas anti-inflammatory agents are described in the PCT Published PatentApplication, WO 98/56771.

None of the above described proposals have resulted in a clinicaldevelopment of an imaging agent for the early diagnosis of Alzheimer'sdisease. Accordingly, there is still a clinical need for a diagnosticagent that could be used for a reliable and early diagnosis ofAlzheimer's disease.

SUMMARY OF THE INVENTION

The present invention is directed to radiopharmaceuticals that bind tothe CCR1 receptor and are able to pass through the blood-brain barrier,and are therefore useful in diagnosing Alzheimer's disease, preferablyat an early stage of the disease.

Accordingly, in one aspect, the invention is directed to compounds offormula (I):

wherein:

-   X¹ and X² are each independently halo;-   R¹ and R² are each independently hydrogen or alkyl; and-   R³ is hydrogen, amino, monoalkylamino, dialkylamino,    monoaralkylamino, alkylcarbonylamino, alkenylcarbonylamino,    haloalkylcarbonylamino, arylcarbonylamino, alkoxyalkylcarbonylamino,    alkoxycarbonylalkylcarbonylamino, glycinamido, monoalkylglycinamido,    arylcarbonylglycinamido, aminocarbonylglycinamido,    (aminocarbonyl)(alkyl)glycinamido, (alkoxyalkylcarbonyl)glycinamido,    ureido, monoalkylureido, monoarylureido, monoaralkylureido, or    alaninamido;-   and wherein either one of X¹ or X² is selected from the group of    ¹²³I, ¹²⁵I, ¹²⁸I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁸⁰Br and ¹⁸F; or wherein one of    the carbon atoms in the compound is ¹¹C;-   or a pharmaceutically acceptable salt thereof.

In another aspect, the invention is directed to compounds of formula(II):

wherein

-   X¹ and X² are each independently halo;-   R¹ and R² are each independently hydrogen or alkyl; and-   R⁴ is hydrogen; and-   R⁵ comprises a chelator capable of binding a radioactive metal atom    chosen from the group of ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re;-   or as a complex with ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re;-   or a pharmaceutically acceptable salt thereof.

In another aspect, this invention is directed to a method of diagnosingAlzheimer's disease in a human which comprises administering to a humanin need of such diagnosis compound of formula (I) or formula (II), asdescribed above and herein, and measuring the radioactivity arising fromthe administration of the compound to the human either by using a gammacamera or by positron emission tomography (PET).

In another aspect, the invention is directed to a method of using acompound of the invention for the manufacture of a radiopharmaceuticalfor the diagnosis of Alzheimer's disease in a human.

In another aspect, the invention is directed to a method of preparingcompounds of the invention.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

Various other features and attendant advantages of the present inventionwill be more fully appreciated as the same becomes better understoodwhen considered in conjunction with the accompanying drawings, in whichlike reference characters designate the same or similar parts throughoutthe several views, and wherein:

FIG. 1 shows the expression of CCR1 in Alzheimer's disease brain tissue.

FIG. 2 shows CCR1 antibody specificity in Alzheimer's disease braintissue.

FIG. 3 shows CCR1-Aβ¹⁻⁴⁰ double-labeled tissue sections

FIG. 4 shows neuritic plaques double-labeled for CCR1 and Aβ¹⁻⁴².

FIG. 5 shows diffuse Aβ¹⁻⁴² staining in Alzheimer's disease brain.

FIG. 6 is a graph showing the relationship between CCR1 and Aβ¹⁻⁴⁰ byCDR score.

FIG. 7 is a graph showing the relationship between CCR1 and Aβ¹⁻⁴² byCDR score.

FIG. 8 is a graph demonstrating the decrease in total brainradioactivity over time.

FIG. 9 is a graph showing the percent of injected dose in brain andplasma.

FIG. 10 are graphs showing CCR1 expression in other neurodegenerativediseases.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification and appended claims, unless specified tothe contrary, the following terms have the meaning indicated:

“Alkyl” refers to a straight or branched chain monovalent or divalentradical consisting solely of carbon and hydrogen, containing nounsaturation and having from one to eight carbon atoms, e.g., methyl,ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), n-heptyl, and the like.

“Alkylcarbonylamino” refers to a radical of the formula —N(H)—C(O)—R_(a)where R_(a) is an alkyl radical as defined above, e.g., acetylamino,ethylcarbonylamino, n-propylcarbonylamino, and the like.

“Alkenyl” refers to a straight or branched chain monovalent or divalentradical consisting solely of carbon and hydrogen, containing at leastone double bond and having from two to eight carbon atoms, e.g.,ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and thelike.

“Alkenylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(c) where R_(c) is an alkenyl radical as defined above,e.g., ethenylcarbonylamino, prop-2-enylcarbonylamino,but-2-enylcarbonylamino, and the like.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above, e.g., methoxy, ethoxy, n-propoxy,1-methylethoxy (iso-propoxy, n-butoxy, n-pentoxy, 1,1-dimethylethoxy(t-butoxy), and the like.

“Alkoxycarbonylalkylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(a)—C(O)OR_(a) where each R_(a) is independently an alkylradical as defined above, e.g., ethoxycarbonylmethylcarbonylamino,methoxycarbonylmethylcarbonylamino,(2-ethoxycarbonylethyl)carbonylamino,(2-methoxycarbonylethyl)carbonylamino, and the like.

“(Alkoxyalkylcarbonyl)glycinamido” refers to a radical of the formula—N(H)(O)—CH₂—N(H)(O)—R_(a)—O—R_(a) where each R_(a) is independently analkyl radical as defined above, e.g., (methoxyacetyl)glycinamido,(ethoxyacetyl)glycinamido, and the like.

“Amino” refers to the radical —NH₂.

“Aminocarbonylglycinamido” refers to a radical of the formula—N(H)C(O)—CH₂—N(H)—C(O)—NH₂.

“(Aminocarbonyl)(alkyl)glycinamido” refers to a radical of the formula—N(H)—C(O)—CH₂—N(R_(a))—C(O)—NH₂ where R_(a) is an alkyl radical asdefined above.

“Aryl” refers to a phenyl or naphthyl radical. Unless stated otherwisespecifically in the specification, the term “aryl” or the prefix “ar-”(such as in “aralkyl”) is meant to include aryl radicals optionallysubstituted by one or more substituents selected from the groupconsisting of halo, alkyl, alkoxy, haloalkyl, haloalkoxy, nitro, amino,monoalkylamino, and dialkylamino, as defined herein.

“Arylcarbonylamino” refers to a radical of the formula —N(H)—C(O)—R_(b)where R_(b) is an aryl radical as defined above, e.g.,(4-methoxyphenyl)carbonylamino, (4-fluorophenyl)carbonylamino,(4-chlorophenyl)carbonylamino, and the like.

“Arylcarbonylglycinamido” refers to a radical of the formula—N(H)—C(O)—CH₂—N(H)—C(O)—R_(b) where R_(b) is an aryl radical as definedabove, e.g., phenylcarbonylglycinamido,(4-fluoro-3-trifluoromethylphenyl)carbonylglycinamido,(4-fluorophenyl)carbonylglycinamido, and the like.

“Aralkyl” refers to a radical of the formula—R_(a)R_(b) where R_(a) isan alkyl radical as defined above and R_(b) is an aryl radical asdefined above, e.g., benzyl, and the like.

“Alkoxyalkylcarbonylamino” refers to a radical of the formula—N(H)—C(O)—R_(a)—O—R_(a) where each R_(a) is an alkyl radical as definedabove, e.g., methoxymethylcarbonylamino, ethoxyethylcarbonylamino,methoxyethylcarbonylamino, and the like.

“Alaninamido” refers to a radical of the formula —N(H)—C(O)—C(CH₃)H—NH₂.

“Benzyl” refers to a radical of the formula —CH₂—R_(h) where R_(h) is aphenyl radical optionally substituted by one or more substituentsselected from the group consisting of halo, alkyl, haloalkyl, alkoxy,nitro, amino, monoalkylamino, and dialkylamino.

“Dialkylamino” refers to a radical of the formula —N(R_(a))R_(a) whereeach R_(a) is independently an alkyl radical as defined above, e.g.,dimethylamino, methylethylamino, diethylamino, dipropylamino,ethylpropylamino, and the like.

“Glycinamido” refers to a radical of the formula —N(H)—C(O)—CH₂—NH₂.

“Halo” refers to bromo, chloro, iodo or fluoro.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2-trifluoroethyl,1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl,1-bromomethyl-2-bromoethyl, and the like.

“Haloalkylcarbonylamino” refers to a radical of the formula—N(H)(O)—R_(f) where R_(f) is an haloalkyl radical as defined above,e.g., trifluoromethylcarbonylamino, trifluoromethylcarbonylamino,2-bromoethylcarbonylamino, and the like.

“Monoalkylamino” refers to a radical of the formula —N(H)R_(a) whereR_(a) is an alkyl radical as defined above, e.g., methylamino,ethylamino, propylamino, and the like.

“Monoaralkylamino” refers to a radical of the formula —N(H)R_(d) whereR_(d) is an aralkyl radical as defined above, e.g., benzylamino,(3,4,5-trimethoxybenzyl)amino, (4-chlorobenzyl)amino, and the like.

“Monoalkylglycinamido” refers to a radical of the formula—N(H)—C(O)—CH₂—N(H)R_(a) where R_(a) is an alkyl radical as definedabove.

“Monoalkylureido” refers to a radical of the formula—N(H)—C(O)—N(H)R_(a) or a radical of the formula —N(R_(a))—C(O)—NH₂where R_(a) is an alkyl radical as defined above.

“Monoarylureido” refers to a radical of the formula —N(H)—C(O)N(H)Rb ora radical of the formula —N(R_(b))—C(O)—NH₂ where R_(b) is an arylradical as defined above.

“Monoaralkylureido” refers to a radical of the formula—N(H)—C(O)—N(H)R_(d) or a radical of the formula —N(R_(d))—C(O)—NH₂where R_(d) is an aralkyl radical as defined above.

“Optional” or “optionally” means that the subsequently described eventof circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid and the like, andorganic acids such as acetic acid, propionic acid, pyruvic acid, maleicacid, malonic acid, succinic acid, fumaric acid, tartaric acid, citricacid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, p-toluenesulfonic acid, salicylic acid, and the like. Particularlypreferred salts of compounds of the invention are the monochloride saltsand the dichloride salts.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, zinc, aluminum salts and the like. Preferred inorganic saltsare the ammonium, sodium, potassium, calcium, and magnesium salts. Saltsderived from organic bases include, but are not limited to, salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

“Ureido” refers to a radical of the formula —N(H)—C(O)—NH₂.

It is understood from the above definitions and examples that forradicals containing a substituted alkyl group any substitution thereoncan occur on any carbon of the alkyl group.

The compounds of the invention, or their pharmaceutically acceptablesalts, may have asymmetric carbon atoms in their structure. Thecompounds of the invention and their pharmaceutically acceptable saltsmay therefore exist as single enantiomers, diastereoisomers, racemates,and mixtures of enantiomers and diastereomers. All such singleenantiomers, diastereoisomers, racemates and mixtures thereof areintended to be within the scope of this invention. Absoluteconfiguration of certain carbon atoms within the compounds, if known,are indicated by the appropriate absolute descriptor R or S.

Separate enantiomers can be prepared through the use of optically activestarting materials and/or intermediates or through the use ofconventional resolution techniques, e.g., enzymatic resolution or chiralHPLC.

Utility and Administration

The compounds of the invention as described herein are antagonists tothe chemokine receptor known as CCR1 and have the ability to pass theblood-brain barrier. The compounds are therefore suited as in vivodiagnostic agents for imaging of Alzheimer's disease. The detection ofradioactivity is performed according to well-known procedures in theart, either by using a gamma camera or by positron emission tomography(PET).

Preferably, the free base or a pharmaceutically acceptable salt form,e.g. a monochloride or dichloride salt, of a compound of the inventionis used in a galenical formulation as diagnostic agent. The galenicalformulation containing the compound of the invention optionally containsadjuvants known in the art, e.g. buffers, sodium chloride, lactic acid,surfactants etc. A sterilization by filtration of the galenicalformulation under sterile conditions prior to usage is possible.

The radioactive dose should be in the range of 1 to 100 mCi, preferably5 to 30 mCi, and most preferably 5 to 20 mCi per application.

Testing

The suitability of the compounds as imaging agents for Alzheimer'sdisease can be demonstrated by experimental protocols known to those ofordinary skill in the art. For example, the upregulation of CCR1receptors in Alzheimer's disease brains can be demonstrated inimmunohistochemical staining experiments of autopsy brain tissuecollected from Alzheimer's disease patients as described in detail belowin the Examples. The ability of the compounds of the invention to bindto the CCR1 receptor and their ability to pass through the blood-brainbarrier, can also be assessed in known in vitro and in vivo assays asdescribed below in the Examples. In particular, Example 10 describes alarge study that was undertaken to address the degree of CCR1 expressionin different stages of Alzheimer's disease. Brain tissue from 50 autopsycases showed a correlation between degree of clinical severity(dementia) in Alzheimer's disease and CCR1 expression in dystrophicneurites. CCR1 expression in plaque-like structures within the brains ofclinically normal individuals is rare. Also, CCR1 expression is notfound in the brains of individuals with other neurodegenerative diseasesunless there is a concomitant Alzheimer's disease pathology(specifically, Aβ¹⁻⁴² in plaques).

Preferred Embodiments

Of the various aspects of the invention, certain compounds of formula(I) are preferred. In particular, compounds of formula (1) wherein X¹ isa chloro at the 4-position of the phenyl ring and X² is a ¹⁸F atom atthe 4-position of the phenyl ring are preferred. Especially preferredare such compounds for use as diagnostic agents in positron emissiontomography (PET).

Even more preferred are those compounds of formula (I) wherein R³ is inthe 2-position of the phenyl ring and R¹ is a methyl at the 2-positionof the piperazinyl ring and R² is a methyl at the 5-position of thepiperazinyl ring. Equally preferred are those compounds of formula (I)wherein R³ is in the 2-position of the phenyl ring and R¹ is a methyl inthe 2-position of the piperazinyl ring and R² is hydrogen.

Further preferred are these preferred compounds in their mono- ordichloride salt form.

Of the various aspects of the invention, certain compounds of formula(II) are preferred. In particular, compounds of formula (II) wherein—N(R⁴)R⁵ is in the 2-position of the phenyl ring and R¹ is in the2-position of the piperazinyl ring and R² is in the 5-position of thepiperazinyl ring. Equally preferred are those compounds of formula (II)wherein —N(R⁴)R⁵ is in the 2-position of the phenyl ring and R² is amethyl in the 2-position of the piperazinyl ring and R² is hydrogen.

Even more preferred are those compounds of formula (II) wherein R⁵comprises a chelator according to formula (III):

or formula (IV):

as well as their complexes with ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re. Of thesepreferred compounds, even more preferred are those compounds of formula(II) wherein R⁵ comprises a chelator according to formula (III) or (IV),and wherein the chelator is bound to the nitrogen in the —N(R⁴)R⁵ groupof the non-radioactive compound of formula (II) via a linker moietycomprising an alkyl radical having one to ten carbon atoms, wherein thealkyl radical optionally contains one to ten —C(O)-groups, one to ten—C(O)N(R)-groups, one to ten —N(R)C(O)-groups, one to ten —N(R)-groups,one to ten —N(R)₂ groups, one to ten hydroxy groups, one to ten —C(O)OR—groups, one to ten oxygen atoms, one to ten sulfur atoms, one to tennitrogen atoms, one to ten halogen atoms, one to ten aryl groups, andone to ten saturated or unsaturated heterocyclic rings wherein R ishydrogen or alkyl. For example, the linker moiety can be an alkylradical having 1 to 10 carbon atoms, wherein one or more CH₂ groups isoptionally replaced by, in each case independently, —C—, —S—, —N(R)—,—C(O)—, —C(O)N(—R)— or N(R)C(O)— groups. This alkyl radical can also beoptionally substituted by one or more —N(R)₂ groups, hydroxy groups,—C(O)OR groups, or halogen atoms, wherein R is hydrogen or alkyl. Apreferred linker moiety is —C(O)—CH₂N(H)—.

Of the compounds of the invention, the most preferred compounds offormula (I) are those compounds selected from the group consisting ofthe following:

-   1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,    technetium-99m-complex;-   1-(2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodo-¹²³I-phenyl)urea;-   N′-(2-mercaptoeth-1-yl)-N′(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,    technetium-99m-complex;-   N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,    technetium-99m-complex;-   2-(2-amino-4-chlorophenoxy)-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-(2-amino-4-chlorophenoxy)-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-(2-amino-4-chlorophenoxy)-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-(2-amino-4-chlorophenoxy)-1-((2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-[4-chloro-2-(diethyramino)phenoxy]-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;-   1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;-   1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;-   1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3(2,4-dichlorophenyl)urea;-   1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{(2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;-   1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;-   1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;-   1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;-   N-(5    chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;-   (E)-N-5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;-   (E)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;-   (E)-N-5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;-   (E)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;-   methyl    N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;-   methyl    N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;-   methyl    N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;-   methyl    N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;-   ethyl    N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;-   ethyl    N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;    ethyl N-(5-chloro-2-{2-[(2SR,5SR)    (4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;-   ethyl    N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;-   N-(5-chloro-2-{2-(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]2-oxoethoxy}phenyl)propanamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)propanamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)propanamide;-   N-(5-chloro-2-{2-((2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)propanamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)3-fluorobenzamide;-   N-(5-chloro-2-{2-((2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;-   N-(5-chloro-2{-2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)3-fluorobenzamide;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;-   1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)3-(p-tolyl)urea;-   1-(5-chloro-2-{2-[(2RS,5RS)-4(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;-   1-(5-chloro-2-{2-[(2SR,    5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;-   1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;-   1-(5-chloro-2-{2-[(2RS,5SR)-4-(4fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;-   1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;-   1-(5-chloro-2-{2-[(2RS,5RS)4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;-   1-(5-chloro-2-{2[(2SR,5RS)-4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;-   1-benzyl-3-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-benzyl-3-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-benzyl-3-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-benzyl-3-(5-chloro-2{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;-   1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,    5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;-   1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;-   1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;-   2-(2-benzylamino-4-chlorophenoxy)-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)2,5-dimethylpiperazin-1-yl)ethan-1-one;-   2-(2-benzylamino-4-chlorophenoxy)-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-(2-benzylamino-4-chlorophenoxy)-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   2-(2-benzylamino-4-chlorophenoxy)-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]2-oxoethoxy}phenyl)glycinamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;-   1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   1-(5-chloro-2-{2-[(2S)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethyl-piperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethyl-piperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;-   2-bromo-N-(5-chloro-2-{(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   2-bromo-N-(5-chloro-2-{(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   2-bromo-N-(5-chloro-2{(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   2-bromo-N-(5-chloro-2-{(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;-   N-(5-chloro-2-{2-[(2RS,5SR)4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;-   N-(5-chloro-2-{2-[(2RS,5SR)4(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;-   (2RS)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   (2SR)-N-(5-chloro-2-{2-(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   (2RS)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   (2SR)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   (2RS)-N-(5chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   (2SR)-N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   (2RS)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   (2SR)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;-   N-(chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2    oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;-   N-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;    and-   N-(5-chloro-2-{2-[(2S)-4-(fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;    as well as the mono- and dichloride salts thereof.

The compounds of the present invention may be administered by anysuitable route, preferably in the form of a pharmaceutical compositionadapted to such a route, and in a dose effective to bind to CCR1receptors in the brain and thereby be detected by gamma camera or PET.Typically, the administration is parenteral, e.g., intravenously,intraperitoneally, subcutaneously, intradermally, or intramuscularly.Intraveneous administration is preferred.

Thus, for example, the invention provides compositions for parenteraladministration which comprise a solution of contrast media dissolved orsuspended in an acceptable carrier, e.g., serum or physiological sodiumchloride solution.

Aqueous carriers include water, alcoholic/aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringersdextrose, etc. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyl oleate.

Other pharmaceutically acceptable carriers, non-toxic excipients,including salts, preservatives, bufers and the like, are described, forinstance, in REMMINGTONS PHARMACEUTICAL SCIENCES, 15^(th) Ed. Easton:Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and THE NATIONALFORMULARY XIV., 14^(th) Ed. Washington: American PharmaceuticalAssociation (1975). Aqueous carriers, are preferred.

Pharmaceutical composition of this invention are produced in a mannerknown per se by suspending or dissolving the compounds of thisinvention—optionally combined with the additives customary in galenicpharmacy—in an aqueous medium and then optionally sterilizing thesuspension or solution. Suitable additives are, for example,physiologically acceptable buffers (such as, for instance,tromethamine), additions of complexing agents (e.g.,diethylenetriaminepentaacetic acid) or—if required—electrolytes, e.g.,sodium chloride or—if necessary—antioxidants, such as ascorbic acid, forexample.

If suspensions or solutions of the compounds of this invention in wateror physiological saline solution are desirable for enteraladministration or other purposes, they are mixed with one or several ofthe auxiliary agents (e.g., methylcellulose, lactose, mannitol) and/ortensides (e.g., lecithins, Tween, Myrj) and/or flavoring agents toimprove taste (e.g., ethereal oils), as customary in galenic pharmacy.

The compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

For the compounds according to the invention having radioactivehalogens, these compounds can be shipped as “hot” compounds, i.e., withthe radioactive halogen in the compound and administered in e.g., aphysiologically acceptable saline solution. In the case of the metalcomplexes, these compounds can be shipped as “cold” compounds, i.e.,without the radioactive ion, and then mixed with Tc-generator eluate orRe-generator eluate.

Preparation of the Compounds of the Invention

A. Preparation of Compounds of Formula (I)

In general, the radioactive imaging agents of formula (I) of the presentinvention are prepared by reacting radioactive 4-halobenzyl derivativeswith piperazine derivatives. Preferred are ¹⁸F-labeled 4-fluorobenzylderivatives for PET-imaging. A general method for the preparation of4-fluoro-¹⁸F-benzyl halides is described in Iwata et al., AppliedRadiation and Isotopes (2000), Vol. 52, pp. 87-92.

The ¹⁸F-labeled 4-fluorobenzyl derivatives are prepared by reaction of abenzaldehyde compound of formula (a) with ¹⁸F⁻ ions to obtain abenzaldehyde compound of formula (b):

wherein LG is a leaving group, for example, bromo, chloro, iodo, nitro,or N(R)₃ ⁺X⁻ (where R is alkyl and X is a halo ion, such as Br⁻, Cl⁻, orI⁻; an ion of a alkanoic acid, such as an acetate ion (CH₃C(O)O⁻); or anion of an alkylsulfonic acid or haloalkylsulfonic acid, such as triflat(CF₃SO₃ ⁻)). Preferably LG is triflat. Starting from the compound offormula (b), several synthetic pathways are possible in preparing thecompounds of formula (I).

In a first synthetic pathway, a compound of formula (b) is reduced withNaBH₄ to obtain a compound of formula (c):

The compound of formula (c) is then reacted with HI, P₂I₄ or Ph₃PBr₂ toobtain an iodo- or bromo-substituted compound of formulae (d) or (e):

The compounds of formulae (d) and (e) can be obtained with aradiochemical yield of 50 to 60%. The radiochemical purity is greaterthan 95%. The specific activity of the compounds is 5 mCi per 1 nmol.

A compound of formulae (d) or (e) can then be reacted with a piperazinederivative (f) to obtain a compound of formula (g) (a compound offormula (I)):

Compounds of formula (f) may be prepared according to methods known tothose of ordinary skill in the art and is described in detail in PCTPublished Patent Application, WO 98/56771.

In a second synthetic pathway, a compound of formula (b) is directlyreacted with a compound of formula (f) using a reducing agent, forexample, formic acid, ammonium formiate, NaBH₄, or NaBH₃CN, to obtain acompound of formula (g) (a compound of formula (I)):

B. Preparation of Compounds of Formula (II)

For Single Photon Emission Computed Tomography (“SPECT”),^(99m)Tc-labeled compounds are preferred. Those compounds are compoundsof formula (II). A general synthetic pathway for these compounds startswith non-radioactive analogues of compounds of formula (II) that arereacted with ^(99m)Tc-binding chelators, e.g. N₂S2-Chelators.Preparation of the non-radioactive analogs of the compounds of formula(II) is described in detail in PCT Published Patent Application, WO98/56771. The synthesis of the chelators follows standard procedures,for example, the procedures described in A. Mahmood et al., A N₂S₂-Tetradentate Chelate for Solid-Phase Synthesis: Technetium, Rheniumin Chemistry and Nuclear Medicine (1999), Vol. 5, p. 71, or in Z. P.Zhuang et al., Bioconjugate Chemistry (1999), Vol. 10, p. 159.

Preferred chelators are chelators of formulae (III) or (IV):

One of the chelators is either bound directly to the nitrogen in the—N(R⁴)R⁵ group of the non-radioactive compound of formula (II), or via alinker moiety comprising an alkyl radical having one to ten carbonatoms, wherein the alkyl radical optionally contains one to ten—C(O)-groups, one to ten —C(O)N(R)-groups, one to ten —N(R)C(O)-groups,one to ten —N(R)-groups, one to ten —N(R)₂ groups, one to ten hydroxygroups, one to ten —C(O)OR— groups, one to ten oxygen atoms, one to tensulfur atoms, one to ten nitrogen atoms, one to ten halogen atoms, oneto ten aryl groups, and one to ten saturated or unsaturated heterocyclicrings wherein R is hydrogen or alkyl. A preferred linker moiety is—C(O)CH_(2—N(H)—.)

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following specific examples are provided as aguide to assist in the practice of the invention, and are not intendedas a limitation on the scope of the invention.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and, all parts and percentages areby weight, unless otherwise indicated.

The entire disclosures of all applications, patents and publications,cited above and below, and of corresponding U.S. Provisional ApplicationSer. No. 60/246,299, filed Nov. 6, 2000, are hereby incorporated byreference.

EXAMPLE 1 Preparation of 1-(5-Chloro-2-{2-[(2R)-2-methylpiprazin-1-yl]-2-oxoethoxy}phenyl)urea

A. DIEA (19.10 mL, 110 mmol) was added to a solution of(R)-(−)-2-methylpiperazine (10 g, 100 mmol) in 250 mL of methylenechloride. The solution was cooled to −10° C. The BOC anhydride wasdissolved in 250 mL of methylene chloride and this solution was added tothe chilled piperazine solution over 1 hour. The reaction was allowed towarm to ambient temperature over 16 hours. The reaction mixture wasfiltered to remove the solids and the filtrate washed with 500 mL ofwater, dried over magnesium sulfate, filtered and evaporated to an oil.The oil was purified by flash column chromatography to afford 11.0 g ofthe compound of formula (a).

B. The compound of formula (a) (11.0 g, 55 mmol) and DIEA (10.5 mL, 60.4mmol) were dissolved in 100 mL of methylene chloride. The resultingsolution was chilled to −10° C. Chloroacetyl chloride (4.37 mL, 55 mmol)was added dropwise to the solution maintaining the temperature at −10°C. After stirring for 1 hour the reaction mixture was washed with 100 mLof water, dried over magnesium sulfate, filtered and evaporated to anoil. The oil was purified by flash column chromatography to afford 14.8g of the compound of formula (b).

C. To a solution of the compound of formula (b) (14.8 g, 53.5 mmol) andthe compound of formula (e) (9.98 g, 53.5 mmol) in 75 mL of DMSO wasadded potassium carbonate (18.48 g, 133.7 mmol). The resulting mixturewas heated to 50° C. for 3 hours. The mixture was cooled to 30° C. andpoured into 700 mL of water. The water was extracted three times with200 mL of ethyl acetate. The ethyl acetate extracts were combined andwashed with 200 mL of 1 N KOH followed by brine. The organic layer wasthe dried over magnesium sulfate, filtered and evaporated to a foam. Thefoam was purified by flash column chromatography to afford 19.6 g of thecompound of formula (c).

D. The compound of formula (c) (7.68 g, 18 mmol) was dissolved in 40 mLof ethyl acetate. The resulting solution was chilled in an ice bath andanhydrous HCl gas was bubbled through the solution for 5 minutes. Theproduct precipitated while the mixture was allowed to sit at ambienttemperature for 1 hour. The product was collected by filtration, washedon the filter with fresh ethyl acetate, and dried under vacuum atambient temperature to constant weight to afford 5.9 g of1-(5-chloro-2-{2-[(2R)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea,the compound of formula (d), as white solid; NMR (2 rotomers); (400 MHz,DMSO) 9.7 (m, 0.5H), 9.2 (m, 0.5H), 8.16 (s, 1H), 8.13 (s, 0.5H), 6.8(s, 2H), 4.9 (m, 2H), 4.4 (m, 5H), 3.8 (bs, 0.5H), 3.4, (bs, 0.5H), 3.2,(m, 2.5H), 3.0 (m, 2 H) 1.2-1.4 (m, 3H) ppm.

EXAMPLE 2 Preparation of the Compound of Formula (e)

To a solution of 2-amino-4-chlorophenol (10 g, 69.7 mmol) in 100 mL ofanhydrous THF at ambient temperature was added trimethylsilyl isocyanate(18.8 mL, 139.4 mmol) in one portion. The solution was heated to 60° C.and remained at this temperature for 22 hours at which time water (1.3mL, 76.7 mmol) was added. After 30 minutes the solution was cooled toambient temperature and concentrated to a brown oil. This oil wasdissolved in ethyl acetate, treated with activated carbon, dried overmagnesium sulfate and filtered. The filtrate was concentrated to a pinksolid which was crystallized from 10:1, toluene/methanol to give 5.4 gof the compound of formula (e) as tan powder.

EXAMPLE 3 Preparation of1-(5-Chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea

A. Hydrogen fluoride-¹⁸F was prepared in a cyclotron by bombardment ofH₂O—¹⁸O with protons. The resulting hydrogen fluoride-¹⁸F was adsorbedon an anion-exchange cartridge. The hydrogen fluoride-¹⁸F was elutedwith a solution of Kryptofix 222 (15 mg, 40 μmol) and K₂CO₃ (2.77 mg, 20μmol) in aqueous acetonitrile (1.5 mL, 66%). The radioactive fractionswere evaporated to dryness in a nitrogen gas stream. This procedure wasrepeated three times with dry acetonitrile (1 mL). After addition of asolution of 4-trimethylammonium-benzaldehyde-triflate (2 mg, 6.4 μmol)in dry DMF (250 μl) the resulting reaction mixture was heated for 5minutes to 100° C. After cooling to ambient temperature a solution of1-(5-chloro-2-{2-((2R)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea,a compound of formula (d), (3 mg, 8.3 μmol) in 50 μl acetic acid and asolution of sodium-cyano-borhydride (4 mg, 63.7 μmol) in 100 μl dry DMFwere added. The reaction mixture was heated to 120° C. for 10 minutes.After addition of 5 mL of water the mixture was filtered over apolystyrol-cartridge. The adsorbed product was washed with 2 mL of waterto afford the title compound,1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea,which was eluted with 1.5 mL acetonitrile and purified by HPLC.

B. In a similar manner, other compounds of formula (I) containing a ¹⁸Fatom are prepared.

EXAMPLE 4 Preparation ofN-(5-Chloro-2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide

A. To a solution of (R)-(−)-2-methylpiperazine (2.0 g, 20 mmol) in 20 mLCH₂Cl₂ was added DIEA (5.29, 40 mmol) and 4-fluorobenzyl chloride (2.39mL 20 mmol). The resulting mixture was stirred at ambient temperaturefor 15 hours. After the reaction was completed, the reaction mixture waswashed with water (3×20 mL) and brine, then dried over Na₂SO₄, filteredand concentrated in vacuo to afford the compound of formula (f) (2.2 g)as a white solid.

B. To a solution of the compound of formula (f) (2.2 g, 10 mmol) in 50mL CH₂Cl₂ was added chloroacetyl chloride (0.84 mL, 10 mmol). Theresulting mixture was stirred at ambient temperature for 10 minutes andthen triethylamine (3 mL, 21 mmol) was added. After 30 minutes, themixture was washed with water (3×20 mL) and brine, then dried overNa₂SO₄, filtered and concentrated in vacuo to afford an oil.Purification by flash column chromatography afforded the compound offormula (g) (2.5 g).

C. To a solution of the compound of formula (g) (2.4 g, 8.4 mmol) in 50mL DMF was added K₂CO₃ (2.5 g, 17 mmol), NaI (0.2 g) and4-chloro-2-nitrophenol (1.3 g, 8.4 mmol). The resulting mixture washeated at 70-80° C. After 1 hour, the mixture was concentrated in vacuo,then taken up in ethyl acetate (150 mL) and washed with water (3×100mL)and then brine. The organic layer was separated, dried over Na₂SO₄,filtered and concentrated in vacuo to afford an oil. Purification byflash column chromatography afforded the compound of formula (h) (3.1g).

D. To a solution of the compound of formula (h) (1.1 g, 2.6 mmol) in 10mL ethanol was added a solution of tin(II) chloride dihydrate (3.0 g, 13mmol) in 5 mL ethanol. The resulting mixture was heated at 75° C. After1 hour, the reaction was concentrated in vacuo, then taken in ethylacetate (100 mL), washed with 1 N NaOH solution in water (3×100 mL) andbrine. The organic layer was separated, dried over Na₂SO₄, filtered andconcentrated in vacuo to afford an oil. Purification by flash columnchromatography afforded the compound of formula (i) (0.75 g).

E. To a solution of the compound of formula (i) (0.7 g, 1.78 mmol) in 10mL DMF was added BOC-Gly-OSU (0.58 g, 2.13 mmol). The resulting mixturewas heated at 50-60° C. After 24 hours, the mixture was concentrated invacuo, then taken up in ethyl acetate (150 mL) and washed with water(3×100 mL) and then brine. The organic layer was separated, dried overNa₂SO₄, filtered and concentrated in vacuo to afford an oil.Purification by flash column chromatography afforded the compound offormula (j) (0.7 g).

F. To a solution of the compound of formula (j) (0.6 g, 1.1 mmol) in 10mL CH₂Cl₂ was added TFA (5 mL). The resulting mixture was heated atambient temperature. After the reaction was completed in 1 hour, thereaction was concentrated in vacuo, then taken up in ethyl acetate (100mL), washed with 1N NaOH solution in water (2×100 mL) and brine. Theorganic layer was separated, dried over Na₂SO₄, filtered andconcentrated in vacuo to afford an oil. Purification by flash columnchromatography affordedN-(5-chloro-2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide,the compound of formula (k), (0.45 g) as a white solid; NMR (CDCl₃) 10.2(s, 1), 8.5 (s, 1), 7.3 (m, 2), 7.0 (m, 3), 6.8 (d, 1), 4.7 (m, 2),3.4-3.6 (m, 5), 3.0 (m, 1), 2.8 (m, 1), 2.6 (d, 1), 2.2 (m, 1), 2.0 (m,2), 1.2-1.4 (m, 3) ppm.

EXAMPLE 5N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,Technetium-99m-complex

A. To a stirred solution of N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide (175.5 mg, 0.4mmol) (the compound of formula (k) as prepared above in Example 4),N—(S-trityl-2-mercaptoeth-1-yl)-N—(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)glycine (291.4 mg, 0.4mmol) (which can be synthesized according to A. Mahmood et al., A N₂S₂-Tetradentate Chelate for Solid-Phase Synthesis: Technetium, Rheniumin Chemistry and Nuclear Medicine (1999), Vol. 71), andN-hydroxysuccinimide (45.5 mg, 0.4 mmol) in 5 mL dichloromethane wasadded dropwise a solution of dicyclohexylcarbodiimide (81.4 mg, 0.4mmol) in 3 mL dichloromethane. The resulting suspension was stirred overnight at ambient temperature. After filtration, the resulting solutionwas evaporated under reduced pressure. The desired product,N′-(S-trityl-2-sulfanyleth-1-yl)-N′-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide(340 mg, 72.8%) was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 98:2) as a white powder.

Elemental Analysis: Calc.: C 69.93 H 5.87 N 7.20 O 6.85 S 5.49 Found: C69.69 H 6.05 N 7.01 O S 5.32

B.N′-(S-Trityl-2-sulfanyleth-1-yl)-N′-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide(116.8 mg, 0.1 mmol), as prepared above, was dissolved in 5 mLtrifluoroacetic acid. After addition of triethyl silane (48 μl, 0.3mmol) the resulting suspension was stirred for 15 minutes at ambienttemperature. The fitrate was evaporated under reduced pressure and theresidue was triturated with 7 mL diethyl ether. The precipitate wasstirred for 1 hour at ambient temperature and filtered off, yielding thedesired product,N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,bis-trifluoracetic acid salt (87 mg, 95.5%), as a white solid.

Elemental Analysis: Calc.: C 44.81 H 4.65 N 9.22 O 15.80 S 7.04 Found: C44.54 H 4.91 N 8.99 O S 6.80

C. Disodium tartrate (1 mg) andN′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,bis-trifluoracetic acid salt (100 μg), as prepared above, were dissolvedin 500 μl sodium phosphate buffer (0.1 M, pH=8.5). After addition of37MBq ^(99m)Tc-generator eluate, 5 μl tin-(II) chloride solution wasadded and the mixture was heated for 10 min to 100° C. HPLC analysisshowed a major peak indicating that the desired product,N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,Technetium-99m-complex, was synthesized with a RCP>90%.

EXAMPLE 6 Preparation of1-(2-{2-[(2R)-4-(4-Fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodophenyl)urea

A. To a solution of p-iodophenol (2.2 g, 10 mmol) in 18 mL acetic acidwas added a solution of HNO₃ (0.7 mL, 70%) in 5 mL acetic acid dropwiseover 10 minutes. The resulting mixture was stirred at ambienttemperature. After 30 minutes, the reaction mixture was diluted by 100mL ice-water. The precipitate was collected, washed with 100 mL water.Purification by flash column chromatography afforded 1.2 g of2-nitro-4-iodophenol.

B. To a solution of the compound of formula (g) (0.3 g, 1.05 mmol) (asprepared herein) in 10 mL DMF was added K₂CO₃ (0.45 g, 3.2 mmol), NaI(0.01 g) and 2-nitro-4-iodophenol (0.28 g, 1.05 mmol). The resultingmixture was heated at 70-80° C. After 1 hour, the mixture wasconcentrated in vacuo, then taken up in ethyl acetate (150 mL) andwashed with water (3×100 mL)and then brine. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated in vacuo toafford an oil. Purification by flash column chromatography afforded 0.28of the compound of formula (l).

C. To a solution of the compound of formula (l) (0.28 g, 0.55 mmol) in 5mL ethanol was added a solution of Tin(II) chloride dehydrate (0.616 g,2.73 mmol) in 5 mL ethanol. The resulting mixture was heated at 75° C.After 1 hour, the reaction was concentrated in vacuo, then taken up inethyl acetate (100 mL), washed with 1N NaOH solution in water (3×100 mL)and brine. The organic layer was separated, dried over Na₂SO₄, filteredand concentrated in vacuo to afford an oil. Purification by flash columnchromatography afforded the compound of formula (m) (0.25 g).

D. To a solution of the compound of formula (m) (0.25 g, 0.52 mmol) in 3mL AcOH was added water (6 mL). The resulting mixture was stirred atambient temperature for 10 minutes, then a solution of KOCN (0.085 g,1.0 mmol) in 1 mL water was added dropwise. The reaction mixture wasstirred at ambient temperature for 10 minutes, then heated at 55° C. for5 minutes. After the reaction was completed, the reaction mixture wasconcentrated in vacuo, then taken up in CH₂Cl₂ (50 mL), washed with 2NNaOH solution in water (2×100 mL) and brine. The organic layer wasseparated, dried over Na₂SO₄, filtered and concentrated in vacuo toafford an oil. Purification by flash column chromatography afforded thecompound of formula (n) (0.16 g) as a white solid; NMR (CDCl₃) 9.0 (s,1), 8.6 (s, 1), 7.3 (m, 2), 7.0 (t, 3), 6.6 (d, 1), 4.9 (s, 2), 4.7 (m,2), 4.4 (m, 1), 3.4-3.6 (m, 3), 3.0 (m, 1) 2.8 (m, 1), 2.6 (d, 1), 2.2(m, 1), 2.0 (m, 1), 1.2-1.4 (m, 3) ppm.

EXAMPLE 7 Preparation of1-(2-{2-[(2R)-4-(4-Fluorobenzyl)-2-methylpiperazin-1-y]-2-oxoethoxy}-5-iodo-¹²³I-phenyl)urea

A. To a solution of the compound of formula (n) (1 mg), as preparedabove, and 10 μg copper-(II)-sulfate in 300 μl DMF was added 1 mCisodium iodine[¹²³I] solution. The resulting reaction mixture was heatedover night to 100° C. After adding of 1 mL half-saturated aqueous NaHCO₃solution the product was extracted with 2 mL CH₂Cl₂. The organic layerwas evaporated to dryness in a nitrogen gas stream. The desired product,1-(2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodo-¹²³I-phenyl)urea,was purified using a RP-cartridge [eluant: EtOH/water (2:1)].

B. In a similar manner as described above, other compounds of formula(I) are prepared.

EXAMPLE 8

N′-(2-Mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,Technetium-99m-complex

A. To a stirred solution of 156.7 mg (0.4 mmol)5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]aniline(compound of formula (i)) (291.4 mg, 0.4 mmol),N—(S-trityl-2-mercaptoeth-1-yl)-N—(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)glycine(synthesized according to A. Mahmood et at., A N ₂ S ₂-TetradentateChelate for Solid-Phase Synthesis: Technetium, Rhenium in Chemistry andNuclear Medicine (1999), Vol. 5, p. 71) and N-hydroxysuccinimide (45.5mg, 0.4 mmol) in 5 mL dichloromethane was added dropwise a solution ofdicyclohexylcarbodiimide (81.4 mg, 0.4 mmol) in 3 mL dichloromethane.The resulting suspension was stirred over night at ambient temperature.After filtration, the resulting solution was evaporated under reducedpressure. The desired product,N′-(S-trityl-2-mercaptoeth-1-yl)-N′—(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide(352 mg, 79.2%), was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 98:2) as a white powder.

Elemental Analysis: Calc.: C 71.36 H 5.90 N 6.30 O 5.76 S 5.77 Found: C71.08 H 6.13 N 6.05 O S 5.52

B.N′-(S-trityl-2-mercaptoeth-1-yl)-N′-(S-trityl-5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]-phen-1-yl}glycinamide(111.1 mg, 0.1 mmol) was dissolved in 5 mL trifluoroacetic acid. Afteraddition of 48 pl (0.3 mmol) triethyl silane the resulting suspensionwas stirred for 15 min at ambient temperature. The fitrate wasevaporated under reduced pressure and the residue was triturated with 7mL diethyl ether. The precipitate was stirred for 1 hour at ambienttemperature and filtered off, yielding the desired product,N-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,bis-trifluoracetic acid salt (75 mg, 87.8%) as a white solid.

Elemental Analysis: Calc.: C 44.99 H 4.60 N 8.20 O 14.98 S 7.51 Found: C44.71 H 4.89 N 8.03 O S 7.22

C. Disodium tartrate (1 mg) disodium tartrate andN′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide, bis-trifluoracetic acid salt(100 μg) were dissolved in 500 μl sodium phosphate buffer (0.1M,pH=8.5). After addition of 37.5 MBq ^(99m)Tc-generator eluate, 5 μltin-(II) chloride solution was added and the mixture was heated for 10minutes to 100° C. HPLC-analysis showed a major peak indicating that thedesired product,N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,technetium-99m-complex was synthesized with a RCP>91%.

EXAMPLE 9

N′-(2-Mercaptoeth-1-yl)-N′-(5-mercapto-3azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,Technetium-99m-complex

A. To a stirred solution of5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]aniline(156.7 mg, 0.4 mmol) and triethylamine (40.5 mg, 0.4 mmol) in 5 mLdichloromethane was added dropwise a solution of α-bromoacetyl chloride(63 mg, 0.4 mmol) in 3 mL dichloromethane. The resulting solution wasstirred over night at ambient temperature. The solvent was evaporatedunder reduced pressure. The desired product,N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}2bromoacetamide(175 mg, 85.3%), was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 99:1) as a white powder.

Elemental Analysis: Calc.: C 51.53 H 4.72 N 8.19 O 5.76 Found: C 51.28 H4.99 N 7.92 O

B. A stirred solution ofN-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}-2-bromoacetamide(165 mg, 0.42 mmol) andN,N′-bis-(S-(4-methoxybenzyl)-2-mercaptoeth-1-yl)ethylenediamine (841.3mg, 2 mmol) (synthesized according to Z. P. Zhuang et al., BioconjugateChem. (1999), Vol. 10, p. 159) in 1,4-dioxane (5 mL) was heated underreflux for 24 hours. The resulting reaction mixture was evaporated underreduced pressure. The residue was dissolved in 15 mL dichloromethane andwashed with saturated aqueous sodium carbonate solution. After dryingover MgSO₄ the solvent was evaporated under reduced pressure. Thedesired product,N′-(S-(4-methoxybenzyl)-2-mercaptoeth-1-yl)-N′-(S-(4′-methoxybenzyl)-5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-(4-fluorobenzyl)-2-(2R)methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide(154 mg, 43%), was isolated after silica gel chromatography (eluent:dichloromethane/methanol, 9:1) as a white powder.

Elemental Analysis: Calc. C 61.99 H 6.50 N 8.22 O 9.38 S 7.52 Found: C61.71 H 6.58 N 8.03 O S 7.28

C.N′-(S-(4-methoxybenzyl)-2-mercaptoeth-1-yl)-N′-(S-(4′-methoxybenzyl)-5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide(140 mg, 0.164 mmol) was dissolved at 0° C. in 5 mL trifluoroaceticacid. After addition of Hg(OAc)₂ (104.5 mg, 0.328 mmol) the resultingmixture was stirred for 30 minutes at 0° C. and saturated for 15 minwith H₂S. After filtration the solvent was evaporated under reducedpressure. The resulting yellow oil was triturated with 3 mL diethylether. The desired product,N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-(2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,tris-trifluoracetic acid salt (126 mg (80.5%), was isolated afterfiltration as a white powder.

Elemental Analysis: Calc.: C 42.79 H 4.44 N 7.34 O 15.09 S 6.72 Found: C42.48 H 4.73 N 7.02 O S 6.76

D. Disodium tartrate (1 mg) andN′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,tris-trifluoracetic acid salt (100 μg) were dissolved in 500 μl sodiumphosphate buffer (0.1M, pH=8.5). After addition of 37.5 MBq^(99m)Tc-generator eluate, 5 μl tin-(II) chloride solution was added andthe mixture was heated for 10 minutes to 100° C. HPLC-analysis showed amajor peak indicating that the desired product,N′-(2-mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide.Technetium-99m-complex, was synthesized with a RCP>95%.

EXAMPLE 10 Immunohistochemical Localization of CCR1 in Human Brains

Materials and Methods

A first group of tissue samples from brains that had been obtained atautopsy within 12 hours of death was used for immunohistochemicalstaining of the chemokine receptor CCR1 in Alzheimer's disease andcontrol brains. Paraffin-embedded, 5-μm-thick, unstained sections fromfrontal cortex and hippocampus were used for all immunohistochemical andhistochemical stains.

Slides were de-paraffinized in xylene and hydrated to phosphate-bufferedsaline containing 0.005% Triton X100 (PBS-T). For light microscopy(using DAB as chromogen) endogenous peroxidase activity was blocked byincubating the slides with 0.01% H₂O₂ in methanol for 30 minutes.Non-specific binding was reduced by blocking in 10% normal goat serum(NGS) in PBS for 30 minutes. The primary antibodies [Rabbit anti-CCR1,(N-terminal peptide); Mouse anti-Neurofilament; mouse anti-GFAP; mouseanti-Tau (AT8); mouse anti-CD68 (clone KP1); mouse anti-β-amyloid(Boehringer Mannheim, #1 381 431); Rabbit anti-CCR8, (N-terminalpeptide)] were diluted in PBS-T and slides were incubated overnight atroom temperature. Staining was completed using a Biogenex ABC™ kit. Allwashes used PBS-T. After the DAB reaction was completed, slides werelightly counterstained with Gill's Hematoxylin, dehydrated andcoverslipped with Permount. Before coverslipping, some slides werere-stained with antibodies against Aβ peptide using identical methodsexcept that the chromogen was True Blue™. For immunofluorescentdouble-labeling studies the slides were de-paraffinized, blocked with10% NGS, and incubated with a cocktail of both primary antibodiesovernight. The slides were washed with PBS-T and then incubated with acocktail of goat secondary antibodies, each at 1/50 dilution. To avoidconfusion from endogenous (yellow-green) tissue fluorescence, thesecondary antibodies were conjugated to either Cy3 (ε_(max)=565 nm;goat-anti-mouse, Amersham) or Cy5 (ε_(max)=700 nm; goat-anti-rabbit,Amersham). Slides were viewed on a confocal microscope with akrypton-argon laser (model 2010, Molecular Dynamics, Sunnyvale, Calif.).

Subsequently, a second group of brain tissues composed of 10 cases fromcognitively normal elderly and 40 cases from Alzheimer's diseasepatients who had been assessed for clinical dementia rating (CDR) wereobtained and evaluated for immunohistochemical expression of CCR1. Thesamples were stained for CCR1 and other markers as described aboveexcept that monoclonal antibodies (clone #6D5) specific for Aβ¹⁻⁴² (amarker for diffuse, early amyloid deposits as well as for more matureneuritic plaques) were obtained from Dr. Ursula Moenning. Theseantibodies were visualized with Vector Red™ (Vector Labs). Dr Moenningalso provided monoclonal antibodies specific for Aβ¹⁻⁴⁰ (clone #13E9), amarker for plaques found in late-stage disease, that was visualized withTrue Blue™.

Results

In both sets of brains, areas with Alzheimer's disease pathology showeda characteristic staining pattern. CCR1 immunoreactivity was found inassociation with neuritic (i.e., senile) plaques in both the hippocampalformation and the cerebral cortex. The immunostained structures wereround to ovoid and were not usually associated with cell bodies. Theyvaried in size and appeared to be filled with a punctate granularmaterial. These structures were found to form “coronas” around theamyloid deposits in senile plaques, but were distinct from the Aβ itself(FIGS. 14). The top panel in FIG. 1 shows a neuritic plaque with acorona of CCR1-positive processes. Neuronal cell bodies of some CA1 andCA3 neurons in Alzheimer's disease cases were sometimes stained by CCR1antibodies. The bottom panel in FIG. 1 illustrates this finding. Thisstaining was distinct from neurofibrillary tangles (NFT) orgranulovacuolar bodies, although it was commonly present in the somataof neurons with these degenerative changes.

Pre-incubation of antibodies with a 16-mer polypeptide from theextracellular (N-terminus) region of the CCR1 receptor proteincompletely blocked all tissue staining, as illustrated in FIG. 2. Inparticular, the top image in FIG. 2 shows a lack of staining inAlzheimer's disease brain with CCR1 antibodies that were pre-incubatedwith the polypeptide while the bottom image in FIG. 2 shows many CCR1-positive structures in a sister section stained with un-incubatedantibodies.

Double-labeling studies showed that nearly all CCR1-positive structureswere associated with neuritic plaques containing Aβ¹⁻⁴² as shown in FIG.4. Aβ¹⁻⁴² in diffuse plaques (FIG. 5) was not typically associated withCCR1 nor with any significant cellular responses. In more advanced casesof Alzheimer's disease, many more Aβ¹⁻⁴⁰-positive plaques were seen.Some, but not all of these were associated with CCR1 staining as shownin FIG. 3. In some cases CCR1-positive plaques were completely free ofAβ¹⁻⁴⁰ staining.

In cases of severe Alzheimer's disease where significant neuronal lossand gliosis were seen, reactive astrocytes in the subiculum andentorhinal cortex were also CCR1 positive. In less severe cases,CCR1-positive reactive astrocytes were uncommon.

Confocal microscopy of double-labeled sections showed that CCR1immunoreactivity co-localized with neurofilament-positive processes. Themacrophage/microglial marker, CD68, was not associated with CCR1staining, nor was the AT8 antibody against abnormally phosphorylated tauprotein. As expected from light microscopic studies, CCR1immunoreactivity did co-localize with GFAP in areas of astrogliosis.

Leukocytes present within cerebral vessels were strongly CCR1 positivein both Alzheimer's disease and control brain tissues, serving aspositive internal controls for staining methods. In FIG. 5 note theCCR1-positive staining of two intravascular cells (arrows). SomeCCR1-positive material is also seen at asterisk, but in general, diffuseplaques are not associated with CCR1.

Using hippocampal tissue from the second study (where patients weregrouped into known clinical categories by CDR score), a quantitativeevaluation of the number of CCR1-positive plaque-like structures in thehippocampus was undertaken. Histologic evaluations of individualsections were performed under blinded conditions with respect toclinical (CDR) status. The volume analyses were conducted using unbiasedcomputerized methods.

FIG. 3 demonstrates the histologic relationship between CCR1-positivedystrophic neurites (brown stain) and a neuritic plaque containingAβ¹⁻⁴⁰ (blue stain). Note in that in FIG. 3 the CCR1-positive processesare distinct from the amyloid. Plaques containing Aβ¹⁻⁴⁰ were discreteand easily counted, as were the clusters of CCR1-positive neurites.Slides of hippocampus were evaluated for the number of CCR1-positivecoronas, the number of Aβ¹⁻⁴⁰ positive plaques, and the number ofplaques containing both markers. The structures were counted by region(e.g., CA3, CA1, and subiculum) within the entire hippocampal formation,including entorhinal cortex and then totaled. The values are relativeestimates of the number of structures in the hippocampus as representedby 5-μm thick cross-sections of the hippocampal formation. FIG. 6 showsthe relationship between CCR1 and Aβ¹⁻⁴⁰ by CDR score.

Quantification of the amount of Aβ¹⁻⁴² required a different techniquebecause Aβ¹⁻⁴² was more abundant and formed “diffuse” plaques that couldnot be easily enumerated (see FIG. 5). For these slides acomputer-assisted method (C.A.S.T. stereology system) was used toestimate the area of entorhinal cortex occupied by Aβ¹⁻⁴². This area wasexpressed as a percent of the total area of cortex on the slide. Theareas occupied by neuritic and diffuse plaques were counted separately.The computer-driven microscope stage assured random (unbiased)evaluation of the tissue sections. The volume % of entorhinal cortexoccupied by Aβ¹⁻⁴² (both diffuse and neuritic) is compared to the numberof CCR1-positive plaques in a sister section of entorhinal cortex inFIG. 7.

FIG. 6 shows that the average number of CCR1-positive dystrophicneurites in the hippocampus increases as a function of CDR score inAlzheimer's disease. The number of positive structures in earlyAlzheimer's disease (CDR 0.5) is increased above control (COR 0) levels.Although the differences between control and Alzheimer's disease groupsdid not become statistically significant until group CDR 2, theseexpression patterns support the conclusion that CCR1 is upregulated indystrophic neuronal processes even at very early stages of Alzheimer'sdisease. Note that the number of Aβ¹⁻⁴⁰ plaques do not rise until latein the disease. CCR1 expression in brain tissue may thus be considered arelatively early indicator of Alzheimer's disease.

The correlation between number of CCR1-positive plaques in entorhinalcortex and the amount of Aβ¹⁻⁴² is shown in FIG. 7. In general, CCR1levels in entorhinal cortex rise as the disease state increases;however, the area sampled is much smaller than the area sampled in FIG.6. Note, however, that Aβ¹⁻⁴² levels rise early in the disease.

EXAMPLE 11 Assessment of Brain Availability, Using a ¹⁴C-Labeled Tracer

A ¹⁴C analogue of1-(5-chloro-2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)ureawas prepared and used for pharmacokinetic studies of brain availability.Mice were injected i.v. through cannulated jugular veins with theanalogue at 36 mg/kg containing 334,000 dpm per dose. Mice (in groups offour) were sacrificed at 1, 15, 30, 120, and 1440 minutes afterinjection by CO₂ inhalation followed by intra-cardiac puncture forremoval of whole blood. The chest was then opened and mice were perfusedthrough the heart with phosphate buffered saline for five minutes toremove residual radioactive drug from the blood compartment. The brainswere then removed, weighed, and sampled for amount of radioactivity intwo separate pieces of cerebral cortex. The radioactivity levels per mgof tissue in the two samples were averaged and the data were normalizedto total brain weight, as illustrated in FIG. 8. The bars represent thestandard error (n=4 mice per time point). In particular, the graph inFIG. 8 shows that the total number of disintegrations per minute (DPM)for ¹⁴C analogue in whole mouse brain decreases to negligible levels bytwo hours after injection. At the 1-minute time point, calculations showthat, on average, about 3% of the injected dose is found in whole mousebrain (average weight of 450 g).

Plasma samples prepared from the whole blood removed from the mice werealso analyzed for radioactive content. The amount of radioactivity inequal volumes of brain and plasma was compared over time as a percent ofinjected dose, as illustrated in FIG. 9. The % of injected dose (i.d.)found per mL of plasma also declines over time, reaching negligiblelevels after two hours. For comparison to brain levels, the total numberof DPM per brain were normalized to a gram of brain weight and expressedas a percentage of the i.d. per gram of brain tissue. Note that mousebrains weigh on average about 450 mg so that the % of i.d. in 1 g ofmouse brain shown in the graph overestimates the real value by abouttwo-fold. It is clear that the levels of the analogue in the brain fallin concert with plasma levels. Although the analogue is lipophilic,normal mouse brain does not appear to be a depot site for the CCR1antagonist.

EXAMPLE 12 CCR1 Expression in Other Neurodegenerative Diseases

Materials and Methods

Histologic samples of autopsy brain tissue from a total of 29 cases fromseven different neurodegenerative diseases (other than pure Alzheimer'sdisease) were obtained and studied for CCR1 content. Slides wereimmunostained with antibodies against CCR1 as described in Example 10above and reviewed under blinded conditions with respect to the specificneurodegenerative disease. Subsequently, sister sections weredouble-labeled with antibodies against CCR1 (DAB as chromogen) andAβ¹⁻⁴² (Vector Red™ as chromogen). The diseases examined are listedbelow:

Parkinson's disease (PD) 6 cases Parkinsonian dementia of Guam 3 casesCongophilic angiopathy 4 cases Multi-infarct dementia (MID) 4 casesDiffuse Lewy body dementia (DLBD) 4 cases Pick's disease 4 casesProgressive supranuclear palsy (PSP) 4 cases

The stained slides were graded for CCR1 and Aβ¹⁻⁴² content using ahistologic scale of 0 to 4, with 0 being absence of staining, 0.5indicating rare expression, and grades 1 through 4 indicating increasinglevels of expression with 4 being highly abundant.

Results

All six cases of Parkinson's disease and all three cases of Parkinsoniandementia of Guam were negative for CCR1. CCR1-positive plaque-likestructures, similar to those found in Alzheimer's disease, were observedin all 4 cases of congophilic angiopathy, in 3 of the 4 cases of DLBD,in 2 out of the 4 cases of PSP, and in 1 out of the 4 cases of both MIDand Pick's disease.

Double-labeling studies confirmed the assumption that theseCCR1-positive plaque-like structures were associated with Aβ¹⁻⁴², as wasfound in pure cases of Alzheimer's disease (Example 10). FIG. 10 showsthe results of the pathological evaluations in graphic form for all thediseases except for Parkinson's (which was negative for CCR1 in brain).Basically, CCR1 expression was never found in brain tissue samplesunless Aβ¹⁻⁴²-positive neuritic plaques were also present.

After the code was broken, it was found that the one case of Pick'sdisease showing CCR1 expression also carried the diagnosis ofAlzheimer's disease. The two PSP cases with CCR1 were also diagnosedwith concurrent Alzheimer's disease. Congophilic angiopathy and DLBD arediseases that are closely associated with Alzheimer's disease pathology.Therefore, it is not surprising that they would also have high levels ofCCR1 expression in association with Aβ¹⁻⁴². In elderly populations it isoften the case that Alzheimer's disease pathology will overlay otherdisease processes. This was found in one of the four MID cases. Theseresults suggests that CCR1 is a marker that is closely associated withAlzheimer's disease pathology (specifically, Aβ¹⁻⁴²-positive neuriticplaques) regardless of other concurrent pathological processes that maybe present in brain. As such, it is likely to be highly specific forAlzheimer's disease pathology and therefore may be useful as adiagnostic surrogate marker of disease progression.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of diagnosing Alzheimer's disease in a human patient whichcomprises: administering to a patient in need of such diagnosis acompound according to of formula (I):

wherein: X¹ and X² are each independently halo; R¹ and R² are eachindependently hydrogen or alkyl; and R³ is hydrogen, amino,monoalkylamino, dialkylamino, monoaralkylamino, alkylcarbonylanmino,alkenylcarbonylamino, haloalkylcarbonylamino, arylcarbonylamino,alkoxyalkylcarbonylamino, alkoxycarbonylalkylcarbonylamino, glycinamido,monoalkylglycinamido, arylcarbonylglycinamido, aminocarbonylglycinamido,(aminocarbonyl)(alkyl)glycinamido, (alkoxyalkylcarbonyl)glycinamido,ureido, monoalkylureido, monoarylureido, monoaralkylureido, oralaninamido; and wherein either one of X¹ or X² is selected from thegroup of ¹²³I, ¹²⁵I, ¹²⁸I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁸⁰Br and ¹⁸F; or whereinone of the carbon atoms in the compound is ¹¹C; or a pharmaceuticallyacceptable salt thereof, and measuring the radioactivity arising fromthe administration of the compound to said patient either by using agamma camera or by positron emission tomography (PET).
 2. A method ofdiagnosing Alzheimer's disease in a human patient which comprisesadministering to said patient in need of such diagnosis a compoundaccording to formula (II):

wherein X¹ and X² are each independently halo; R¹ and R² are eachindependently hydrogen or alkyl; and R⁴ is hydrogen; and R⁵ is a grouphaving a radical containing a chelator capable of binding a radioactivemetal atom selected from the group of ^(99m)Te, ¹⁸⁶Re and ¹⁸⁸Re; or as acomplex with ^(99m)Tc, ¹⁸⁶Re and ¹⁸⁸Re; or a pharmaceutically acceptablesalt thereof; and measuring the radioactivity arising from theadministration of the compound to said patient either by using a gammacamera or by position emission tomography (PET).
 3. A method accordingto claim 1, wherein said compound binds to chemokine reseptor CCR1 andpasses the blood-brain barrier.
 4. A method according to claim 2,wherein said compound binds to chemokine receptor CCR1 and passes theblood-brain barrier.
 5. A method according to claim 1, wherein R¹ ismethyl at the 2-position of the piperazinyl radical and R² is methyl atthe 5-position of the piperazinyl radical.
 6. A method according toclaim 2, wherein R¹ is methyl at the 2-position of the piperazinylradical and R² is methyl at the 5-position of the piperazinyl radical.7. A method according to claim 1, wherein R¹ is methyl at the 2-positionof the piperazinyl radical and R² is hydrogen.
 8. A method according toclaim 2, wherein R¹ is methyl at the 2-position of the piperazinylradical and R² is hydrogen.
 9. A method of claim 1, wherein X¹ is chloroat the 4-position of the phenyl radical and X² is a ¹⁸F atom at the4-position of the phenyl radical.
 10. A method of claim 2, wherein saidchelator is of formula (III):


11. A method of claim 2, wherein said chelator is of formula (IV):


12. A method of claim 10, wherein in group R⁵ a linker moiety is presentbetween said chelator and the reminder of the compound.
 13. A method ofclaim 11, wherein in group R⁵ a linker moiety is present between saidchelator and the reminder of the compound.
 14. A method of claim 12,wherein the linker moiety is —C(O)—CH₂N(H).
 15. A method of claim 13,wherein the linker moiety is —C(O)—CH₂—N(H).
 16. A method according toclaim 1, wherein said compound is a monochloride salt.
 17. A methodaccording to claim 2, wherein said compound is a monochloride salt. 18.A method according to claim 1, wherein said compound is a dichloridesalt.
 19. A method according to claim 2, wherein said compound is adichloride salt.
 20. A method according to claim 1, wherein X¹ and X²are each independently bromo, chloro, iodo or fluoro; R¹ and R² are eachindependently hydrogen, methyl, ethyl, n-propyl, 1-methylethyl, n-butyl,1,1-dimethylethyl, n-pentyl, or n-heptyl; and R³ is hydrogen, amino,methylamino, ethylamino, propylamino, dimethylamino, methylethylamino,diethylamino, dipropylamino, ethylpropylamino, benzylamino,(3,4,5-trimethoxybenzyl)amino, (4-chlorobenzyl)amino, acetylamino,ethylcarbonylamino, n-propylcarbonylamino, ethenylcarbonylamino,prop-2-enylcarbonylamino, but-2-enylcarbonylamino,trifluoromethylcarbonylamino, trifluoromethylcarbonylamino,2-bromoethylcarbonylamino, (4-methoxyphenyl)carbonylamino,(4-fluorophenyl)carbonylamino, (4-chlorophenyl)carbonylamino,alkoxyalkylcarbonylamino wherein the alkoxy and alkyl portion each have1 to 8 carbon atoms, ethoxycarbonylmethylcarbonylamino,methoxycarbonylmethylcarbonylamino,(2-ethoxycarbonylethyl)carbonylamino,(2-methoxycarbonylethyl)carbonylamino, glycinamido,—N(H)—C(O)—CH₂—N(H)R_(a), phenylcarbonylglycinamido,(4-fluoro-3-trifluoromethylphenyl)carbonylglycinamido,(4-fluorophenyl)carbonylglycinamido, aminocarbonylglycinamido,—N(H)—C(O)—CH₂—N(R_(a))—C(O)—NH₂, (methoxyacetyl)glycinamido,(ethoxyacetyl)glycinamido, ureido, —N(H)—C(O)—N(H)R_(a),—N(R_(a))—C(O)—NH₂—N(H)—C(O)—N(H)R_(b), —N(R_(b))—C(O)—NH₂,—N(H)—C(O)—N(H)R_(d), —N(R_(d))—C(O)—NH₂, or alaninamido; R_(a) is analkyl radical having from one to eight carbon atoms; R_(b) is a phenylor naphthyl radical which is optionally substituted by one or moresubstituents selected from the group consisting of bromo, chloro, iodoor fluoro, alkyl having from one to eight carbon atoms, alkoxy havingfrom one to eight carbon atoms, haloalkyl having from one to eightcarbon atoms, haloalkoxy having from one to eight carbon atoms, nitro,amino, —N(H)R_(a), and —N(R_(a))R_(a) where each R_(a) is independentlyalkyl having from one to eight carbon atoms, and R_(d) is an aralkylgroup in which the alkyl portion has one to eight carbon atoms and thearyl portion is R_(b); wherein either one of X¹ or X² is selected fromthe group of ¹²³I; ¹²⁵I, ¹²⁸I, ¹³¹I, ⁷⁵Br, ⁷⁶Br, ⁸⁰Br and ¹⁸F or one ofthe carbon atoms in the compound is ¹¹C.
 21. A method according to claim1, wherein X¹ and X² are each independently bromo, chloro, iodo orfluoro; and R¹ and R² are each independently hydrogen, methyl, ethyl,n-propyl, 1-methylethyl, n-butyl, 1,1-dimethylethyl, n-pentyl, orn-heptyl.
 22. A method according to claim 2, wherein in group R⁵ alinker moiety is present between said chelator and the reminder of thecompound, wherein the linker moiety is an alkyl radical having one toten carbon atoms, wherein the alkyl radical optionally contains one toten —C(O)-groups, one to ten —C(O)N(R)-groups, one to ten—N(R)C(O)-groups, one to ten —N(R)-groups, one to ten —N(R)₂ groups, oneto ten hydroxy groups, one to ten —C(O)OR-groups, one to ten oxygenatoms, one to ten sulfur atoms, one to ten nitrogen atoms, one to tenhalogen atoms, and one to ten aryl groups.
 23. A method according toclaim 10, wherein in group R⁵ a linker moiety is present between saidchelator and the reminder of the compound, wherein the linker moiety isan alkyl radical having one to ten carbon atoms, wherein the alkylradical optionally contains one to ten —C(O)—groups, one to ten—C(O)N(R)-groups, one to ten —N(R)C(O)—groups, one to ten —N(R)—groups,one to ten —N(R)₂ groups, one to ten hydroxy groups, one to ten—C(O)OR-groups, one to ten oxygen atoms, one to ten sulfur atoms, one toten nitrogen atoms, one to ten halogen atoms, and one to ten arylgroups.
 24. A method according to claim 11, wherein in group R⁵ a linkermoiety is present between said chelator and the reminder of thecompound, wherein the linker moiety is an alkyl radical having one toten carbon atoms, wherein the alkyl radical optionally contains one toten —C(O)—groups, one to ten —C(O)N(R)-groups, one to ten—N(R)C(O)—groups, one to ten —N(R)-groups, one to ten —N(R)₂ groups, oneto ten hydroxy groups, one to ten —C(O)OR-groups, one to ton oxygenatoms, one to ton sulfur atoms, one to ten nitrogen atoms, one to tenhalogen atoms and one to ten aryl groups.
 25. A method according toclaim 1 wherein the radioactive dose administered to said patient is 1to 100 mCi per application.
 26. A method according to claim 2, whereinthe radioactive dose administered to said patient is 1 to 100 mCi perapplication.
 27. A method according to claim 3, wherein the radioactivedose administered to said patient is 1 to 100 mCi per application.
 28. Amethod according to claim 4, wherein the radioactive dose administeredto said patient is 1 to 100 mCi per application.
 29. A method accordingto claim 1, wherein said compound is selected from the group consistingof the following:1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(2-{2-[(2R)-4-(4-fluorobenzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}-5-iodo-¹²³I-phenyl)urea;2-(2-amino-4-chlorophenoxy)-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-(2-amino-4-chlorophenoxy)-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-(2-amino-4-chlorophenoxy)-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-(2-amino-4-chlorophenoxy)-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-[4-chloro-2-(diethylamino)phenoxy]-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(2,4-dichlorophenyl)urea;1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea; 1-(5-chloro-2-{2[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl])-2,5-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-(2-isopentylamino-4-chlorophenoxy)ethan-1-one;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-methylpropanamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamideN-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamideN-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamideN-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxy)acetamide;(E)-N-5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;(E)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;(E)-N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin1-yl]-2-oxoethoxy}phenyl)-2-butenamide;(E)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-butenamide;methylN-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;methylN-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;methylN-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;methylN-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin1-yl]-2-oxoethoxy}phenyl)succinamate; ethylN-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin1-yl]-2-oxoethoxy}phenyl)succinamate; ethylN-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;ethylN-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;ethylN-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)succinamate;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;N-5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)propanamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)propanamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)propanamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)propanamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;N-(5-chloro-2-{2[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-yl]-2-oxoethoxy}phenyl)-3-fluorobenzamide;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(p-tolyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-ethylurea;1-benzyl-3-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-benzyl-3-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-benzyl-3-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-benzyl-3-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;1-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;1-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;1-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;1-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-3-(4-nitrophenyl)urea;2-(2-benzylamino-4-chlorophenoxy)-1-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-(2-benzylamino-4-chlorophenoxy)-1-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-(2-benzylamino-4-chlorophenoxy)-1-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;2-(2-benzylamino-4-chlorophenoxy)-1-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]ethan-1-one;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;1-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea,1-(5-chloro-2-{2[(2S)-(4-fluoro-¹⁸F-benzyl)-2,5-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)urea;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methylamino)acetamide;2-bromo-N-(5-chloro-2-{(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;2-bromo-N-(5-chloro-2-{(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;2-bromo-N-(5-chloro-2-{(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;2-bromo-N-(5-chloro-2-{(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(ureido)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(1-methylureido)acetamide;(2RS)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;(2SR)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;(2SR)-N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;(2SR)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;(2SR)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;(2SR)-N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;(2RS)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;(2SR)-N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-aminopropanamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2,4-difluorobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;N-(5-chloro-2-{2-[(2RS,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;N-(5-chloro-2-{2-[(2SR,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;N-(5-chloro-2-{2-[(2RS,5SR)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(methoxyacetylamino)acetamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2SR,5RS)-4-(4-fluoro-¹⁸F-benzyl)-2,5-dimethylpiperazin-1-yl]-2-oxoethoxy}phenyl)-2-(2-iodobenzoylamino)acetamide;N-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;N-(5-chloro-2-{2-[(2R)-4-(4-fluoro-¹⁸F-benzyl)-2-methylpiperazin-1-yl]-2-oxoethoxy}phenyl)glycinamide;and mono- and dichloride salts thereof.
 30. A method according to claim1, wherein said compound is selected from the group consisting of thefollowing:N′-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycylglycinamide,technetium-99m-complex;N′(2-(mercaptoeth-1-yl)-N′-(5-mercapto-3-aza-2-oxopent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,technetium-99m-complex;N′-(2-(mercaptoeth-1-yl)-N′-(5-mercapto-3-azapent-1-yl)-N-{5-chloro-2-[2-[4-(4-fluorobenzyl)-2-(2R)-methylpiperazin-1-yl]-2-oxoethoxy]phen-1-yl}glycinamide,technetium-99m-complex, and mono-and dichloride salts thereof.